Compositions and methods for diagnosing and treating urinary tract infections

ABSTRACT

The present invention relates to methods and compositions for treating urinary tract infections. In particular, the present invention relates to vaccines and immune modulators for treating urinary tract infections.

This application claims priority to provisional application 60/970,661,filed Sep. 7, 2007, which is herein incorporated by reference in itsentirety.

This invention was made with government support under grant number 2 R01AI043363 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatingurinary tract infections. In particular, the present invention relatesto vaccines and immune modulators for treating urinary tract infections.

BACKGROUND OF THE INVENTION

A urinary tract infection (UTI) is an infection that begins in theurinary system. Serious consequences can occur if the infection spreadsto the kidneys. Women are most at risk of developing a UTI. In fact,half of all women will develop a UTI during their lifetimes, and manywill experience more than one. When treated promptly and properly, UTIsrarely lead to complications. But left untreated, a urinary tractinfection can become something more serious than a set of uncomfortablesymptoms. Untreated UTIs can lead to acute or chronic kidney infections(pyelonephritis), which could permanently damage kidneys. Young childrenand older adults are at the greatest risk of kidney damage due to UTIsbecause their symptoms are often overlooked or mistaken for otherconditions. Women who have UTIs while pregnant may also have anincreased risk of delivering low birth weight or premature infants.

UTIs are generally treated with antibiotics as a first line oftreatment. Drugs most commonly recommended for simple UTIs includeamoxicillin (Amoxil, Trimox), nitrofurantoin (Furadantin, Macrodantin),trimethoprim (Proloprim) and the antibiotic combination of trimethoprimand sulfamethoxazole (Bactrim, Septra). For severe UTIs, hospitalizationand treatment with intravenous antibiotics may be necessary. However,many antibiotic resistant bacteria are present in the environment,especially in hospital and other health care settings. Thus, additionaltreatments for UTIs are needed.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for treatingurinary tract infections. In particular, the present invention relatesto vaccines and immune modulators for treating urinary tract infections.

For example, in some embodiments, the present invention provides amethod of inducing an immune response, comprising administering acomposition comprising an effective amount of at least a portion of oneor more antigens (e.g., including, but not limited to, ChuA, c2482, Iha,IroN, IutA, and IreA) to a subject under conditions such that thesubject generates an immune response to a bacteria (e.g., E. coli) inthe subjects urinary tract. In some embodiments, the composition furthercomprises an adjuvant (e.g., cholera toxin). In some embodiments, thecholera toxin is crosslinked to the antigen. In some embodiments, atleast a portion is a peptide that corresponds to extracellular loop 7 ofIroN or loop 6 of IutA. In some embodiments, the immune responseprotects the subject from developing symptoms of a urinary tractinfection. In some embodiments, the subject exhibits decreased levels ofbacteria in their bladder or kidney.

The present invention further provides a method of preventing urinarytract infections in a subject, comprising administering a compositioncomprising an effective amount of one or more antigens (e.g., including,but not limited to, ChuA, c2482, Iha, IroN, IutA, and IreA) to a subjectunder conditions such that the subject does not develop symptoms of aurinary tract infection.

The present invention additionally provides vaccine compositionscomprising the antigens described herein, as well as kit comprising thevaccine and any other components necessary, sufficient or useful foradministering the vaccine (e.g., including, but not limited to,administration devices (e.g., needles, etc.), instructions, sanitationcomponents, temperature components, adjuvants and the like).

Additional embodiments of the invention are described in the belowdescription and accompanying drawings.

DESCRIPTION OF THE FIGURES

FIG. 1 shows colony forming units of E. coli following treatment with(A) 30 μg or (B) 100 μg of purified antigen (Ag) crosslinked to choleratoxin (CT), followed by two 10 μg boosts at one-week intervals.

FIG. 2 shows recombinant antigen purification.

FIG. 3 shows the intranasal immunization schedule.

FIG. 4 shows urinary tract colonization following immunization andchallenge with (A) Hma, (B) IutA, (C) IreA, (D) Iha, (E) combinationsand (F) IroN and IutA peptides.

FIG. 5 shows cytokine responses of splenocytes from Hma vaccinated mice.(a) c2482 cytokine transcript profile. (b) c2482 cytokine secretion.

FIG. 6 shows antigen specific antibody response of Hma vaccinated mice.(a) c2482 serum antibody profile. (b) c2482 secreted antibody.

FIG. 7 shows cytokines secreted by splenocytes from Iha and IreAvaccinated mice. (a) Iha cytokine and secretion (b) IreA cytokinesecretion.

FIG. 8 shows antigen-antibody responses of IreA vaccinated mice. (a)IreA serum antibody profile. (b) IreA secreted antibody.

FIG. 9 shows cytokines secreted by splenocytes from peptide vaccinatedmice. (a) IroN cytokine secretion and (b) IutA cytokine secretion.

FIG. 10 shows antigen specific antibody responses of peptide vaccinatedmice.

FIG. 11 shows gene expression of vaccine candidate antigens in E. colistrains during an active UTI.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

As used herein, the term “peptide” refers to a polymer of two or moreamino acids joined via peptide bonds or modified peptide bonds. As usedherein, the term “dipeptides” refers to a polymer of two amino acidsjoined via a peptide or modified peptide bond.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified”, “mutant”, and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions with its variousligands and/or substrates.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, antigens are purified byremoval of contaminating proteins. The removal of contaminants resultsin an increase in the percent of antigen (e.g., antigen of the presentinvention) in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabelled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. As usedherein, the term “sample” is used in its broadest sense. In one sense itcan refer to a tissue sample. In another sense, it is meant to include aspecimen or culture obtained from any source, as well as biological.Biological samples may be obtained from animals (including humans) andencompass fluids, solids, tissues, and gases. Biological samplesinclude, but are not limited to blood products, such as plasma, serumand the like. These examples are not to be construed as limiting thesample types applicable to the present invention. A sample suspected ofcontaining a human chromosome or sequences associated with a humanchromosome may comprise a cell, chromosomes isolated from a cell (e.g.,a spread of metaphase chromosomes), genomic DNA (in solution or bound toa solid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like. A sample suspectedof containing a protein may comprise a cell, a portion of a tissue, anextract containing one or more proteins and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treatingurinary tract infections. In particular, the present invention relatesto vaccines and immune modulators for treating urinary tract infections.

Uropathogenic Escherichia coli (UPEC) are responsible for the majorityof uncomplicated urinary tract infections, which can present clinicallyas cystitis or pyelonephritis. UPEC strain CFT073, isolated from theblood of a patient with acute pyelonephritis, is a highly cytotoxic andvirulent strain. Experiments conducted during the course of developmentof embodiments of the present invention used the genome sequence ofCFT073 to generate microarrays for comparative genomic hybridization(CGH) analysis of uropathogenic and fecal/commensal E. coli isolates.Genomic DNA from seven UPEC (three pyelonephritis, four cystitis)isolates, three fecal/commensal strains including K-12 MG1655 washybridized to the CFT073 microarray. Microarray data were validatedusing annotated K-12 and CFT073 sequences. The CFT073 genome contains5379 genes, CGH analysis revealed that 2820 (52.4%) of these genes werecommon to all 11 E. coli strains, yet only 173 UPEC-specific genes werefound in all UPEC strains by CGH but in none of the fecal/commensalstrains. When the sequence of three additional sequenced UPEC strains(UTI89, 536, F11) and a commensal strain (HS) were added to theanalysis, 131 genes present in all UPEC strains but in nofecal/commensal strains were identified. Ten novel genomic islands (>30kb) were delineated by CGH in addition to the three known pathogenicityislands. These genomic islands comprise 814 kb of the 5231 kb (15.6%)genome, demonstrating the importance of horizontal transfer for UPEC.UPEC strains contain a greater number of iron acquisition systems thanfecal/commensal strains, reflective of adaptation to the iron-limitingurinary tract environment. Each strain displayed distinct differences inthe number and type of known virulence factors.

Further experiments conducted during the course of development ofembodiments of the present invention identified a series of antigens,for example, that when conjugated to an adjuvant (e.g., cholera toxin),showed protection against infection in the kidney and bladder (SeeExperimental section below).

The present invention is not limited to a particular antigen. Exemplaryantigens include, but are not limited to, Iha (iron-regulated genehomolog adhesin), IreA (iron-responsive element), IVIAT proteins, Hma(c2482 (novel heme-binding protein)), and cytoplasmic proteinsupregulated in urine. In other embodiments, the antigen is ChuA, IroN,or IutA. In some embodiments, the proteins identified in Example 1 areutilized. Additional antigens are known to those of skill in the art. Insome embodiments, one or more antigens are used in combination. Thepresent invention is not limited to a particular combination ofantigens. In some embodiments, one or more, two or more, three or more,or a larger number of antigens are administered in combination.

In some embodiments, fragments of antigens are utilized forimmunization. For example, in some embodiments, extracellular domain orloops (e.g., loop 7 of IroN or loop 6 of IutA) are utilized as antigens.

In some embodiments, antigens are conjugated to adjuvants or otherimmune system modulators. In one exemplary embodiment, antigens areconjugated to cholera toxin. Additional adjuvants and immune systemmodulators are known to those of skill in the art.

An effective amount of the present vaccine is one in which a sufficientimmunological response to the vaccine is raised to protect a subjectexposed to bacteria in the urinary tract (e.g., E. coli) fromcontracting a UTI. Preferably, the subject is protected to an extent inwhich from one to all of the adverse physiological symptoms or effects(e.g., excess bacteria in the urinary tract, inflammation, and pain) ofthe disease to be prevented are found to be significantly reduced.

Preparation of Vaccines which Contain Protein Sequences as ActiveIngredients is Generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines. Theprotein sequences may be formulated into the vaccine as neutral or saltforms known in the art.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated. The composition can be administered in a single dose, or inrepeated doses. Dosages may contain, for example, from 1 to 1,000micrograms of antigen (vaccine), but preferably do not contain an amountof bacterial-based antigen sufficient to result in an adverse reactionor physiological symptoms of infection. Methods are known in the art fordetermining suitable dosages of active antigenic agent.

The vaccine may be given in a single dose schedule or using amultiple-dose schedule. A multiple-dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand/or reinforce the immune response, for example, at 1 day to 1 yearfor a second dose and if needed, a subsequent dose(s) after intervals ofapproximately 1 day to 1 year.

The composition containing the present vaccine may be administered inconjunction with an adjuvant or with an acceptable carrier which mayprolong or sustain the immunological response in the host animal. Anadjuvant is a substance that increases the immunological response to thepresent vaccine when combined therewith. The adjuvant may beadministered at the same time and at the same site as the vaccine or ata different time, for example, as a booster. Adjuvants also mayadvantageously be administered to the animal in a manner or at a site orlocation different from the manner, site or location in which thevaccine is administered. Adjuvants include aluminum hydroxide, aluminumpotassium sulfate, heat-labile or heat-stable enterotoxin isolated fromEscherichia coli, cholera toxin or the B subunit thereof, diphtheriatoxin, tetanus toxin, pertussis toxin, Freund's incomplete adjuvant,Freund's complete adjuvant, and the like. Toxin-based adjuvants, such asdiphtheria toxin, cholera tetanus toxin and pertussis toxin, may beinactivated prior to use, for example, by treatment with formaldehyde.Other possibilities involve the use of immunomodulating substances suchas lymphokines (e.g. IFN-γ, IL-2 and IL-12) or synthetic IFN-gamma.inducers such as poly I:C in combination with the above-mentionedadjuvants.

In some embodiments, the present vaccine composition is administereddirectly to a subject not yet exposed to a bacterium which causes a UTI.In other embodiments, the vaccine is administered to a subject alreadyexhibiting symptoms of a UTI. The present vaccine may be administered,for example, orally, nasally, or parenterally. Examples of parenteralroutes of administration include intradermal, intramuscular,intravenous, intraperitoneal, subcutaneous and intranasal routes ofadministration.

When administered as a solution, the present vaccine may be prepared inthe form of an aqueous solution, a syrup, an elixir, or a tincture. Suchformulations are known in the art, and are prepared by dissolution ofthe antigen and other appropriate additives in the appropriate solventsystems. Such solvents include water, saline, ethanol, ethylene glycol,glycerol, Al fluid, etc. Suitable additives known in the art includecertified dyes, flavors, sweeteners, and antimicrobial preservatives,such as thimerosal (sodium ethylmercurithiosalicylate). Such solutionsmay be stabilized, for example, by addition of partially hydrolyzedgelatin, sorbitol, or cell culture medium, and may be buffered bymethods known in the art, using reagents known in the art, such assodium hydrogen phosphate, sodium dihydrogen phosphate, potassiumhydrogen phosphate and/or potassium dihydrogen phosphate.

Liquid formulations may also include suspensions and emulsions. Thepreparation of suspensions, for example using a colloid mill, andemulsions, for example using a homogenizer, is known in the art.

Parenteral dosage forms, designed for injection into body fluid systems,require proper isotonicity and pH buffering to the corresponding levelsof body fluids. Parenteral formulations are generally sterilized priorto use.

Isotonicity can be adjusted with sodium chloride and other salts asneeded. Other solvents, such as ethanol or propylene glycol, can be usedto increase solubility of ingredients of the composition and stabilityof the solution. Further additives which can be used in the presentformulation include dextrose, conventional antioxidants and conventionalchelating agents, such as ethylenediamine tetraacetic acid (EDTA).

The present invention further provides kits comprising the vaccinecompositions comprised herein. In some embodiments, the kit includes allof the components necessary, sufficient or useful for administering thevaccine. For example, in some embodiments, the kits comprise devices foradministering the vaccine (e.g., needles or other injection devices),temperature control components (e.g., refrigeration or other coolingcomponents), sanitation components (e.g., alcohol swabs for sanitizingthe site of injection) and instructions for administering the vaccine.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Identification of Virulence Genes

This Example describes the identification of E. coli genes associatewith urinary tract infections.

A. Materials and Methods

Bacterial Strains.

E. coli CFT073 was isolated from the blood and urine of a patientadmitted to the University of Maryland Medical System for the treatmentof acute pyelonephritis. This strain is highly virulent in the CBA mousemodel of ascending UTI (Mobley et al., 1993. Molecular Microbiology10:143-55), is cytotoxic for cultured human renal proximal tubularepithelial cells (Mobley et al., 1990. Infection & Immunity 58:1281-9)and its genome has been fully sequenced and annotated (Welch et al.,2002. Proceedings of the National Academy of Sciences of the UnitedStates of America 99:17020-4). Three collections of E. coli strainsisolated from humans with appropriate clinical syndromes were used inthis study.

Pyelonephritis strains (CFT204, CFT269, CFT325) were isolated from theurine or blood of patients who were admitted to the University ofMaryland Medical System with acute pyelonephritis (bacteriuria of ≧10⁵CFU/mL, pyuria, fever, and no other source of infection) (Mobley et al.,1993. supra). Cystitis strains (F3, F11, F24, F54) were isolated fromthe urine of women under the age of 30 years with first episodes ofcystitis and bacteriuria of ≧10⁵ cfu/mL (Stapleton et al., 1991. Journalof Infectious Diseases 163:773-9). Fecal/commensal E. coli isolates(EFC4, EFC9) were collected from healthy women aged 20-50 years with nohistory of diarrhea, antibiotic usage or symptomatic urinary tractinfection within the past month (Mobley et al., 1993. supra).Additionally, the laboratory-adapted fecal/commensal E. coli isolateK-12 MG1655 was used as a negative control for CGH microarrayexperiments as the full genome sequence has been determined (Blattner etal., 1997. Science 277:1453-74).

Genome Alignments of E. coli Strains CFT073 and K-12 MG1655.

The full genomes of E. coli CFT073 (90) (GenBank Accession No. AE014075)and E. coli K-12 MG1655 (8) (GenBank Accession No. U00096) weresequentially aligned in ≦20 kb segments using the coliBASE software(Chaudhuri et al., Nucleic Acids Research 32:D296-9). Using agene-by-gene comparison of these two genomes, it was possible toidentify CFT073 genes that are present in K-12 but not annotated aspresent. Genes were classified as present if (i) the same gene wasannotated in both strains, (ii) an orthologous gene was identified inK-12 or (iii) a gene with a high level of nucleotide identity to aCFT073 gene was found in K-12. Genes that were severely truncated ineither strain were not considered present. The findings from thisgene-by-gene comparison between the E. coli CFT073 and E. coli K-12genomes were used to validate the microarray data.

Bioinformatic Screen of the E. coli CFT073 Genome.

Each of the coding sequences for the E. coli CFT073 genome were comparedagainst the coding sequences for the publicly available uropathogenic E.coli genomes (UTI89, 536 and F11) as well as all other commensal anddiarrheagenic E. coli listed in Table 1 using BLAST Score Ratio Analysis(BSR) (Rasko et al., 2005. BMC Bioinformatics 6:2). The comparisons inthis study were performed using the nucleotide sequences for each codingregion instead of the peptide coding regions to allow direct comparisonbetween the microarray studies and the BSR analysis (Peptide comparisonswere also performed and the data matches the nucleotide comparisons).For each of the predicted coding sequences (CDS) of E. coli CFT073, aBLASTN raw score was obtained for the alignment against itself(REF_SCORE) and the most similar CDS (QUE_SCORE) in each of the genomesof in Table 1. These scores were then normalized by dividing theQUE_SCORE obtained for each query genome CDS by the REF_SCORE. CDS witha normalized ratio of <0.4 were considered to be non-homologous andscored as absent in this dataset. A normalized BLAST score ratio of 0.4is generally similar to two CDS being ˜30% identical over their entirelength. A normalized BLAST score ratio >0.8 indicates that the CDS arehighly conserved and were scored as Present in the study. This valuerepresents nucleotide identity of greater than ˜85-90% identity over 90%of the reference sequence indicative of a highly conserved sequence. CDSlabeled as divergent have BSR values between these two extremes andrepresent genes that have diverged but still show a significant level ofsimilarity that they will be identified as homologs and depending on thelocation of the microarray probe within the gene may be identified aspresent or absent in that dataset.

Comparative Genomic Hybridization (CGH) Microarray Analysis.

The E. coli CFT073-specific DNA microarray (NIMBLEGEN Systems Inc.,Madison, Wis.) includes 5379 ORFs from the CFT073 genome sequence (Welchet al., 2002. Proceedings of the National Academy of Sciences of theUnited States of America 99:17020-4). Each ORF is represented on theglass slide by a minimum of 17 unique probe pairs of 24-mer insitu-synthesized oligonucleotides. Each pair consists of a sequenceperfectly matched to the ORF, and another adjacent sequence harbors twomismatched bases for determination of background andcross-hybridization, equating to 190,000 probes per array.

Total genomic DNA from log-phase UPEC and fecal/commensal E. coliisolates was isolated using Genomic-Tip 500/G columns (Qiagen) accordingto the manufacturer's protocol. The DNA concentration was adjusted toapproximately 1 μg/μL and sent to NIMBLEGEN systems for microarrayanalysis using the E. coli CFT073-specific DNA microarray. Genomic DNAwas labelled with a random prime reaction (Selzer et al., 2005. GenesChromosomes Cancer 44:305-19). DNA (1 μg) was mixed with 1 O.D. of5′-Cy3 labelled random nonamer (TriLink Biotechnologies) in 62.5 mMTris-HCl, 6.25 mM MgCl2 and 0.0875% β-mercaptoethanol, denatured at 98°C. for 5 min, chilled on ice, and incubated with 100 units Klenowfragment (NEB) and dNTP mix [6 mM each in TE] for 2 h at 37° C.Reactions were terminated with 0.5 M EDTA (pH 8.0), precipitated withisopropanol, and resuspended in water. A 50-fold amplification wastypically achieved. Labelled genomic DNA was hybridized to arrays in 1×NIMBLEGEN Hybridization Buffer (NIMBLEGEN systems) for 16 hours at 45°C. using a Hybwheel hybridization apparatus (NIMBLEGEN systems) in arotisserie oven. The next morning, arrays were washed with nonstringentwash buffer (6×SSPE, 0.01% [v/v] tween-20) for 2 min, and then twice instringent wash buffer (100 mM MES, 0.1 M NaCl, 0.01% [v/v] tween-20) for5 min, all at 47.5° C. Finally, arrays were washed again innon-stringent wash buffer (1 min) and rinsed twice for 30 sec in0.05×SSC. Arrays were spun dry in a custom centrifuge and stored untilscanned.

Microarrays were scanned at 5 μm resolution using the GENEPIX 4000bscanner 223 (Axon Instruments, Union City Calif.), and pixel intensitieswere extracted using NIMBLESCAN image extraction and analysis software(NimbleGen). Data from all microarray experiments were normalized byNimbleGen using the technique described by Irizarry and colleagues(Irizarry et al., 2003. Biostatistics 4:249-64) and log 2 transformedprior to analysis. The normalized data took into account the signalintensities from every probe (perfect match and mismatcholigonucleotides) for each ORF in the genome. Normalized data wereanalyzed for the presence/absence of annotated open reading frames(ORFs) compared to the E. coli CFT073 reference strain. ORFs withnormalized array values less than 7.9 were considered to be absent fromthe test strain compared to the reference strain, E. coli CFT073. Thecut-off value varies between individual microarray experiments, asnormalization of data from multiple experiments is dependent upon theset of input data. To validate the normalized, log 2 transformedmicroarray data, a gene-by-gene comparison between the E. coli CFT073and E. coli K-12 genomes was conducted using the coliBASE software(Chaudhuri et al., 2004. Nucleic Acids Research 32:D296-9).

Serotyping and Virulence Gene Identification.

All serotyping and virulence gene identification was conducted by theGastroenteric Disease Center at Pennsylvania State University. UsingPCR, strains were tested for the presence of a range of virulence genesassociated with UPEC and other E. coli strains: LT, Heat labile toxin;STa/STb, Heat stable toxin a,b; STX1/STX2, Shiga toxin types 1,2;CNF1/2, Cytotoxic necrotizing factor 1,2; EAE, intimin-gamma; BFP,Bundle forming pili; O157, O antigen type 157; papG allele, P-fimbrialadhesion genes (alleles I, III); SFA, S-fimbrial adhesin; focG,FIC-fimbrial adhesin.

B. Results

Selection of Strains for Comparison to E. coli CFT073.

Seven uropathogenic strains of E. coli (three pyelonephritis, fourcystitis) were selected for detailed genomic comparison to E. coliCFT073, a pyelonephritis strain used widely for the study of UTI.Serotypes and virulence gene profiles were determined for these strainsand for two fecal or commensal E. coli strains (Table 2). UPEC strainswere represented by five O serogroups (O1, O6, O18, O25 and O75), whichare among the six most common UPEC O-serogroups. Direct genomic sequencecomparison was also used for three additional UPEC strains (UTI89, 536,and F11) and the well characterized commensal strain HS.

Validation of Microarray Data and Comparison of E. coli CFT073 with E.coli K-12 MG1655

Genomic DNA from the seven UPEC strains and two fecal or commensalstrains was hybridized to the CFT073 microarray for the purpose ofcomparative genomic hybridization. To validate this technique, thesignal intensities from the microarrays was compared to an evaluation ofwhether genes of K-12 strain MG1655 are present or absent with respectto CFT073 by direct sequence comparison. Genome alignments betweenCFT073 and MG1655 revealed 4025 open reading frames (ORFs) in common,either as orthologous ORFs or coding regions with substantial identityat the nucleotide level. A cut-off value for the normalized microarraydata was established by comparing array data signal intensity to genomealignments. The normalized microarray value that most closelyrepresented the presence or absence of genes in K-12 (see Materials andMethods) was determined. Array data were normalized and log 2transformed prior to analysis. Using the established cut-off value,microarray analysis identified 3878 genes common to both K-12 andCFT073.

Of the 4025 CFT073 ORFs identified in K-12 by genome alignments, 531 ofthese ORFs are not annotated in K-12 (i.e., predicted to encodehypothetical proteins). Microarray data confirmed the presence of 461 ofthese genes (87%) in the K-12 genome sequence. Many of the genes thatare present in K-12, but appeared to be absent by microarray, wereeither truncated genes or contained divergent nucleotide sequences thatwould have affected DNA hybridization to the CGH arrays. The differencein the number of genes shared between K-12 and CFT073 by genomealignment versus array data was 147 genes, indicating that only 2.7% ofthe genes in the array could be misclassified as absent when they arepresent (i.e., false negative results). Thus, 97.3% of genes wereclassified correctly, validating the microarray for determination ofgene content among strains.

Comparative Genomic Hybridization of E. coli CFT073 with Uropathogenicand Fecal or Commensal E. coli Strains

The number of genes that each E. coli strain had in common with CFT073,based upon microarray data, is shown in Table 3. Pyelonephritis andcystitis isolates (UPEC strains) contained similar numbers of CFT073genes whereas the fecal or commensal strains had, on average, 100 fewergenes than the UPEC isolates; the laboratory-adapted fecal or commensalstrain K-12 had approximately 300 fewer genes than the UPEC isolates.Although the UPEC isolates tended to contain more CFT073 genes than thefecal or commensal strains, this difference was not statisticallysignificant. The number of genes that were common to all 11 E. coli(including CFT073 and fecal or commensal) strains was 2820, representing52.4% of the E. coli CFT073 genome.

Genomic Islands Identified in E. coli CFT7073

The 5379 ORFs of CFT073 are classified as present or absent in the threepyelonephritis, four cystitis and three fecal or commensal E. colistrains. The CGH microarray analysis of eight UPEC and three fecal orcommensal strains clearly revealed the presence of thirteen genomicislands of >30 kb in E. coli strain CFT073 (Table 4). Ten islands arenewly delineated and three islands previously described (REFS) wereconfirmed. These large genomic islands comprise 814 kb of the 5231 kb(15.6%) of the CFT073 genome.

A new nomenclature for these presumptive pathogenicity islands has beenproposed based on this analysis (Table 4). Eight of the 13 genomicislands (62%) were associated with a tRNA locus, and the majority ofislands contained a phage integrase, transposase or insertion sequenceat one or both boundaries of the island. The size of the islands rangedfrom 32-123 kb (median size of 54 kb) and 10 of the 13 (77%) islands hadG+C contents that differed from that of CFT073 (50.5%) (90). Seven ofthe genomic islands contained one or more genes with a putative orestablished role in virulence (PAI, ICFT073, PAI IICFT073, PAIIIICFT073, PAI VCFT073, PAI VIICFT073, PAI XICFT073, HPICFT073), whilesix (PAI IVCFT073, PAI VICFT073, PAI VIIICFT073, PAI IXCFT073, PAIXCFT073, PAI XIICFT073) contained no known virulence genes. However, allof the genomic islands contained a high number of ORFs with hypotheticalor putative functions (Table 4), and thus additional virulence factorsare likely to exist. Phage DNA sequence is common in E. coli CFT073;indeed five cryptic prophage genomes have been identified in thisstrain, although they do not contain sufficient genetic information toproduce viable phage (Welch et al, supra). Genomic islands PAI IVCFT073,PAI VICFT073, PAI VIICFT073 and PAI XCFT073 are particularly phage-richregions of sequence. PAI VIIICFT073, PAI IXCFT073 and HPICFT073 areUPEC-specific islands (found in pyelonephritis and cystitis strainsonly) whereas PAI XIICFT073 is pyelonephritis-specific. Strain CFT204has more PAIs in common with CFT073 compared to the other UPEC isolates,indicating a closer evolutionary relationship between these two strains.The presence of these thirteen CFT073 genomic islands in nine othersequenced bacterial strains was examined using coliBASE genomealignments. Eleven of the CFT073 genomic islands are not present in anyof the strains studied. PAI IVCFT073 however, is present in E. coliE2348/69 (EPEC), Salmonella typhi TY2 and Salmonella typhimurium LT2although differences were observed at ORFs c0933, c0944-c0946 andc0967-c0970. ORFs c0963-c0968 of PAI IVCFT073 are inverted in E. coliE2348/69 (EPEC) relative to the CFT073 genome. Otherwise, the gene orderis conserved between strains the in the genomic island regions.HPICFT073 was identified in E. coli O42 (EAEC) and Yersinia pestis CO92,although a minor difference was observed at ORF c2425, and ORFsc2424-c2429 were annotated differently between the strains. In Y. pestisCO92, the corresponding region of sequence from c2424-c2429 in CFT073 isannotated as irp2 and irp1, and the same ORFs have been predicted in E.coli O42 (EAEC) using Glimmer. The irp1 and irp2 genes encodeiron-repressible yersiniabactin biosynthesis proteins, which, along withfyuA (yersiniabactin receptor) are part of the High Pathogenicity Island(HPI) in Yersinia species (78).

UPEC-Specific Genes

Using CGH analysis, 2820 genes that were common to all of the UPEC andfecal or commensal strains studied were identified. To estimate thenumber of these genes that could be considered UPEC357 specific genes,it was investigated how many genes were present in at least a certainnumber of UPEC strains, but not present in any of the fecal or commensalstrains including strain MG1655. In a conservative assessment, therewere 173 UPEC-specific ORFs that were considered present in all eightUPEC strains (including CFT073), but in none of the fecal or commensalstrains.

To determine whether we were approaching a true estimate of the numberof UPEC-specific genes or whether the number would continue to fall,additional strains were included in the analysis, an analysis of threesequenced UPEC strains was included. If it is asked how many of the 173UPEC367 specific genes are also conserved among the three additionalsequenced UPEC strains, UTI89, 536 and F11, but not present in sequencedcommensal strain HS, the answer is 131. Thus, 131 genes are present inall 11 UPEC strains including CFT073 but in none of the fecal orcommensal strains examined.

ORFs with Hypothetical Functions Comprise Half (66/131 Genes) of These372 UPEC-Specific Genes.

The UPEC-specific group also contained seven ORFs predicted to beinvolved in transcriptional regulation, nine comprise ABC transportsystems (12 individual ORFs annotated as being involved in ABCtransport) and the chu gene cluster involved in heme/hemoglobinutilization.

Virulence-Associated Genes in Uropathogenic E. coli Strains.

The prevalence of eleven virulence-associated genes or operons fromCFT073 (sat, picU, tsh, iha, iroN, sitABCD, iucABCD/iutA, chuSA, hlyAand usp) were assessed in the eight UPEC and three fecal or commensalisolates (Table 5). Pyelonephritis strain CFT204 contains eight of theeleven virulence-associated genes and appears most closely related toCFT073 in terms of gene content and presences of PAIs (Table 5). Thepyelonephritis strains contained the most established virulence factors6, cystitis isolates contained a mean of 4 virulence factors andfecal/commensal strains contained a mean of only 1 (with none present inE. coli K-12). With respect to adhesins, nine of the ten strains in thisstudy were shown by PCR to contain papG alleles I or III (Table 2). papGallele I was present in all of the pyelonephritis strains while alleleIII was seen in the majority of cystitis isolates. However, the pap geneclusters showed many genes with borderline or absent array values,indicating sequence divergence at the nucleotide level. The only gene incontrast to this observation is fimH, the fimbrial tip adhesin of type 1fimbriae, which is present in all 10 UPEC and fecal/commensal isolatesstudied by CGH. The bioinformatic/BSR screen of the CFT073 genomeagainst 14 other sequenced E. coli strains revealed that the fimH geneis present in 12/14 strains, with only the two enteroaggregative E. coli(EAEC) strains lacking the entire fim gene cluster.

As many as twelve putative fimbrial gene clusters have been identifiedin CFT073 (81, 90); ten chaperone-usher family fimbriae and two type IVpili. Several of these chaperone-usher pathway fimbrial gene clusterswere found to be UPEC-specific by CGH, including the yad/htr/ecp genes(c0166-c0172) and ORFs c4207-c4214. In each case, the chaperone-ushergenes were the most highly conserved, the adhesive tip protein was theleast conserved and the minor structural subunits showed varying degreesof conservation between strains. The type IV pilin genes c2394 and c2395were present in all four pyelonephritis isolates and one of fourcystitis isolates (F11), but not in fecal/commensal strains by CGH. BSRanalysis revealed that the type IV pilin genes are only present in UPECstrains 536 and F11.

With respect to iron acquisition, the enterobactin gene cluster (ent/fepgenes) was present in all ten E. coli strains analyzed by CGH and all 14sequenced E. coli strains by BSR comparisons. The yersiniabactinreceptor, encoded for by the fyuA gene in Yersinia pestis CO92 (Parkhillet al., 2001. Nature 413:523-7), is 99.9% identical to CFT073 gene c2436at the nucleotide level. The c2436 gene, annotated as a putativepesticin receptor precursor, is present in all seven UPEC isolates butnone of the fecal/commensal strains analyzed by CGH. The BSRbioinformatics screen revealed that gene c2436 is present in the UPECstrains UTI89, 536 and F11, as well as EPEC strain E110019 and EAECstrain 042. The sitABCD operon is an iron transport system in CFT073that was present in all three pyelonephritis isolates, ¾ cystitisisolates and one fecal/commensal strain. UPEC strains UTI89, 536 andF11, plus EAEC strain O42, contain the sitABCD operon while this irontransport system was absent from ten other E. coli strains. The chuS(c4307) and chuA genes (c4308), involved in heme/hemoglobin transportand binding, respectively, are present in all seven UPEC strains (threepyelonephritis and four cystitis isolates) but none of thefecal/commensal strains. The chuSA genes are present in UPEC strainsUTI89, 536, and F11, EHEC strains EDL933 and Sakai, and EAEC strain 42.The chuSA genes are absent from all other E. coli strains examined,including the commensal strain HS and the laboratory-adapted commensalstrain K-12 MG1655.

For capsule synthesis, the kpsMT genes of CFT073 encode the ATP-bindingcassette (ABC) transporter components of the group II capsule gene locus(Bliss et al., Mol Microbiol 21:221-31), have been associated withvirulence in UPEC (28) and were present in a single pyelonephritisstrain (CFT204). The capsule genes in both UPEC and fecal/commensal E.coli were diverse between strains based upon DNA hybridization to thearrays. This indicates that different strains express different capsulartypes. The BSR data also show that at least one of the kpsMT genes wasclassified as divergent or absent in all 14 sequenced E. coli strains.

The autotransporter tsh (also referred to as vat or haemoglobinprotease) (Heimer et al., 2004. Infect Immun 72:593-7) was present inall pyelonephritis isolates and three cystitis isolates (F3, F11, F24).

The uropathogenic specific protein (usp) is encoded by gene c0133 inCFT073 and was identified in one pyelonephritis and cystitis isolate butin none of the fecal/commensal strains. Furthermore, BSR analysissupported previous studies showing the usp gene is UPEC-specific. 445The usp gene was present in all three UPEC strains but none of the EHEC,ETEC, EPEC, REPEC, EAEC or fecal/commensal E. coli isolates.

TABLE 1 Sequenced E. coli strains used for BLAST Score Ratio (BSR)analysis against CFT073 GenBank Accession Strain Disease Number CFT073UPEC AE014075.1 UTI89 UPEC CP000243.1 536 UPEC CP000247.1 F11 UPECAAJU00000000 K-12 MG1655 Lab-adapted Human Commensal U00096.2 HS HumanCommensal AAJY00000000 EDL933 EHEC AE005174.2 Sakai EHEC BA000007.2101-1 EAEC AAMK00000000 O42 EAEC Sanger Center E24377A ETEC AAJZ00000000B7A ETEC AAJT00000000 E22 REPEC AAJV00000000 E110019 EPEC AAJW00000000B171 EPEC AAJX00000000

TABLE 2 Characteristics of E. coli strains used in this study IsolatedpapG papG Source Strain from Serotype cnf1 Allele I* Allele III* SFAfacG Hemolysin Pyelonephritis CFT073 Blood O6:H1 − + − + + +Pyelonephritis CFT204 Urine O6:H1 − + − − − + Pyelonephritis CFT269Urine O1:H7 − + − − − − Pyelonephritis CFT325 Blood O75:H56 − + − − − −Cystitis F3 Urine O18:H7 + − + + − + Cystitis F11 Urine O6:H31 + − + +− + Cystitis F24 Urine O18:H7 + − + + − + Cystitis F54 Urine O25:H4 − +− − − − Fecal EFC4 Feces OM:H32 − + − − − − Fecal EFC9 Feces OM:H21 − −− − − −

TABLE 3 Number of CFT073 genes present in UPEC and fecal/commensal E.coli strains based on CGH microarrays No. CFT073 Genes in Average ±Strain Type Strain Common SD* Pyelonephritis CFT204 4178 4198 ± 37CFT269 4241 CFT325 4176 F3 4245 4189 ± 38 Cystitis F11 4162 F24 4168 F544181 Fecal/ EFC4 4054 Commensal EFC9 4101  4011 ± 118 K-12 3878

TABLE 4 Genomic islands of >30 kb identified in E. 1029 coli CFT073using CGH Is- No. land ORFs Size % Hypo. No. in Island Location^(a)Associated tRNA (kb) GC^(b) Virulence Genes^(c) within Island ORFs^(d)Island Name 1 c0253-c0368 248,670-348,353 Asp tRNA - aspV 100 47 cdtA(c0345) 99 PAI III_(cfto73)* (248,554-248,630) picU (c0350) 2 intT-ogrK909,332-942,273 — 33 50 [prophage DNA] 34 PAI IV_(cfto73)* 3 c1165-c12931,127,702-1,240,752 Ser tRNA - serX 113 49 mchBCDEF (c1227, c1229-c1232)91 PAI V_(cfto73)* (1,241,062-1,241,149) sfa/F1C operon (c1237-c1247)iroNEDCB (c1250-c1254) Antigen 43 precursor (c1273) 4 c1400-c15071,328,014-1,388,952 — 61 51 [prophage DNA] 90 PAI VI_(cfto73)* 5c1518-c1601 1,397,313-1,451,607 — 54 50 [prophage DNA] 61 PAIVII_(cfto73)* sitDCBA (c1597-c1600) 6 c2418-c2437 2,218,378-2,250,547Asn tRNA - asnT 32 57 Yersiniabactin receptor (c2436) 18 HPI_(cfto73) *(61) (2,217,922-2,217,997) 7 c2449-c2475 2,262,986-2,316,987 Asn tRNA -asnW 54 53 23 PAI VIII_(cfto73)* (2,262,749-2,262,824) 8 c2482-c25282,322,324-2,365,868 — 44 50 34 PAI IX_(cfto73)* 9 c3143-c32063,018,710-3,066,517 — 48 49 [prophage DNA] 52 PAI X_(cfto73)* 10c3385-c3410 3,223,002-3,255,067 Met tRNA - mesV 32 53 Secreted proteinHcp (c3391) 19 PAI XI_(cfto73)* (3,222,311-3,222,387) ClpB protein(c3392) 11 c3556-kpsM 3,406,498-3,529,292 Phe tRNA - pheV 123 47 hlyA(c3570) 86 PAI I_(cfto73) (29) (3,406,225-3,406,300) pap operon(c3582-c3593) iha(c3610) sat(c3619) iutA (c3623) iucDCBA (c3624-c3628)Antigen 43 precursor (c3655) kpsTM (c3697-3698) 12 intC-c45814,274,702-4,342,875 SectRNA - selC 68 47 76 PAI XII_(cfto73)*(4,274,308-3,406,300) 13 c5143-c5216 4,919,568-4,971,387 Phe tRNA - pheU52 48 pap_2 operon (c5179-c5189) 53 PAI II_(cfto73) (71)(4,971,585-4,971,660) ^(a)Location of genomic islands relative to the E.coli CFT073 genome; ^(b)% GC is the G + C content of the genomic island;^(c)Established or putative virulence factors of UPEC that are found inE. coli CFT073; ^(d)CFT073 genes annotated in coliBASE; *PresumptivePAIs named in this study.

TABLE 5 Presence of virulence-associated genes in uropathogenic andfecal/1034 commensal E. coli strains Virulence Factor* Strain sat picUtsh iha iroN sif^(a) fyuA iuc^(b) chuSA hlyA usp CFT204 − + + +− + + + + + − CFT269 − − + − − + + − + − − CFT325 + − + + − + + − + − +F3 − − + − + + + − + + − F11 − − + − + + + − + − − F24 − − + − + − + − +− − F54 − − − − − + + − + − + EFC4 − − − + − + − + − − − EFC9 − − − − +− − − − − − K-12 − − − − − − − − − − − *Established or putativevirulence factors of UPEC; ^(a)sitABCD; ^(b)iucABCD/iutA.

Example 2 Vaccination of Mouse Model

This Example describes the urinary tract colonization of immunized CBA/Jmice 48 hr after transurethral challenge with 108 CFU of E. coli CFT073.Mice received primary immunizations of (A) 30 μg or (B) 100 μg ofpurified antigen (Ag) crosslinked to cholera toxin (CT), followed by two10 μg boosts at one-week intervals. Control mice were vaccinated with CTalone. Bars indicate the median and each circle represents CFU/g ofbladder or kidney from an individual mouse. The limit of detection forthis assay is 200 CFU/g (dotted line).

The Results are shown in FIGS. 1A and 1B. In the first trial, 10 miceper group were vaccinated with Iha (iron-regulated gene homologadhesin), IreA (iron-responsive element), and c2482 (novel heme-bindingprotein) at an initial dose of 30 μg of antigen conjugated to choleratoxin. IreA showed significant protection (P=0.02) in the bladder (2 logreduction in CFU). In a subsequent trial, the initial dose was increasedto 100 μg of antigen conjugated to cholera toxin. Protection was shownin the bladder for Iha, IreA, and c2482 and in the kidney for Iha, IreAand c2482.

Example 3 Additional Antigens

This example describes immunization of mice with additional antigens.Each protein was expressed as a fusion with a C-terminal His₆ tag.Proteins were isolated from outer membrane fractions by nickel-NTAcolumn purification under denaturing conditions in the presence of 8.0 Murea. Pooled fractions containing the ˜75 kDa protein of interest weredialyzed at 4° C. to remove the urea and refold the protein intoPBS+0.05% Zwittergent® detergent (Calbiochem). FIG. 2 shows a schematicof the purification scheme and an SDS-PAGE gel of the purified protein.

CBA/J mice received 100 μg of antigen intranasally, followed by two 25μg boosts at one-week intervals. One week after the final boost, micewere transurethrally challenged with 108 CFU of E. coli CFT073.Protection was assessed at two days post-inoculation by culturingurinary tract tissues. All immunizations were administered intranasally(10 μl/nare) and consisted of antigen chemically crosslinked to choleratoxin at a ratio of 10:1 (antigen:adjuvant). A schematic of thevaccination schedule is shown in FIG. 3.

Results are shown in FIGS. 4-11. Vaccination with heme receptor Hma(c2482) or siderophore receptors IreA or IutA resulted in significantlyreduced CFU levels in the urinary tract tissues of infected mice ascompared to cholera toxin alone (FIG. 4 and Table 6).

The cytokine response of splenocytes from Hma-vaccinated mice wasassayed. Splenocytes were harvested on day 23 and restimulated with 1μg/mL Hma (c2482) in vitro for 6 hours before RNA was isolated foranalysis by quantitative real time PCR (qPCR) or 48 hours beforesupernatants were collected for quantitation by two-site enzymeimmunosorbant assay (ELISA). Splenocytes from immunized mice produce thecytokines IFN-γ and IL-17 when re-stimulated with the antigen (FIG. 5).FIG. 7 shows IFN-γ and IL-17 secreted by splenocytes from Iha- andIreA-vaccinated mice. Splenocytes were harvested on days 21 and 23(post-vaccination, pre- and post-challenge) and restimulated with 1μg/mL Iha or IreA in vitro for 48 hours before supernatants werecollected for quantitation by ELISA.

Antibody responses of vaccinated mice were also assayed. Sera and urinewere collected from pre-vaccination (pre) and on day 21 (post). ELISAplates were coated with 5 μg/mL Hma (c2482) and sera was added at a1:128 dilution. Hma-specific antibodies were detected with isotypespecific antibodies conjugated to alkaline phosphatase (AP). Each dotindicates an individual animal and bars represent the median.**P<0.0001, *P<0.01. ELISA plates were coated with 10 μg/mL Hma andpooled mouse urine was added. Hma-specific antibodies were detected withan IgA-specific secondary antibody conjugated to AP. Vaccinated micealso produce increased IgM, IgG2a, IgG1 and IgA (FIG. 6). FIG. 8 showsantigen specific antibody responses of IreA vaccinated mice. Sera andurine were collected pre-vaccination (pre) and on day 21 (post). ELISAplates were coated with 5 μg/mL IreA and mouse sera was added at 1:128dilution. Ire-A-specific antibodies were detected with isotype specificantibodies conjugated to AP. ELISA plates were coated with 10 μg/mL IreAand pooled mouse urine was added. IreA-specific antibodies were detectedwith an IgA-specific secondary antibody conjugated to AP.

Additionally, synthetic 30-mer peptides were generated that correspondto putative extracellular loop 7 of IroN and loop 6 of IutA. Each wasused to immunize CBA/J mice. Splenocytes were harvested on days 21 and23 (post-vaccination, pre- and post-challenge) and restimulated with 10μg/mL IroN peptide or IutA peptide in vitro for 48 hours beforesupernatants were collected for quantitation by ELISA. Protection frominfection was seen in the kidneys of infected animals.

FIG. 11 shows gene expression of Vaccine Candidate Antigens in E. colistrains during an active UTI. Urine was collected from patients visitinga urology clinic for symptoms of UTI. RNA was immediately stabilizedjust after collection. Bacteria (identified as E. coli) were harvestedby centrifugation and RNA was isolated and converted to cDNA. LabeledcDNA was hybridized to an E. coli CFT073 microarray in triplicate.

Together, these data indicate that outer membrane iron receptors of UPECrepresent vaccine targets to protect against UTI.

TABLE 6 Summary of urinary tract colonization levels followingimmunization with indicated vaccine and challenge with E. coli CFT073.Urine Bladder Kidneys median median median Vaccine (n) CFU/ml^(a)P-value^(b) CFU/g^(a) P-value^(b) CFU/g^(a) P-value^(b) Hma (20) 1.16 ×10³ 0.2089 2.91 × 10³ 0.3138 1.00 × 10² 0.0084 CT^(c) (15) 9.90 × 10³3.22 × 10³ 2.54 × 10⁴ IreA (15) 1.23 × 10³ 0.1833 1.00 × 10² 0.0348 1.24× 10⁴ 0.1459 Iha (15) 1.15 × 10⁵ 0.2170 3.75 × 10³ 0.2263 2.26 × 10⁴0.0869 CT (10) 1.81 × 10⁴ 4.55 × 10⁴ 2.30 × 10⁵ IutA (20) 9.98 × 10²0.0598 3.43 × 10³ 0.0088 2.65 × 10⁴ 0.0067 CT (20) 2.49 × 10⁴ 4.02 × 10⁴1.73 × 10⁵ IroN peptide (14) 6.06 × 10⁴ 0.3170 3.82 × 10³ 0.3085 1.76 ×10² 0.0529 IutA peptide (15) 7.13 × 10³ 0.3090 3.97 × 10⁴ 0.1277 3.34 ×10² 0.0782 CT (10) 2.52 × 10⁴ 1.22 × 10⁴ 1.68 × 10⁴ Hma + IreA (10) 1.90× 10⁴ 0.2790 3.65 × 10³ 0.1200 5.11 × 10⁴ 0.3667 Hma + IutA (10) 7.94 ×10⁴ 0.4512 3.15 × 10⁴ 0.2135 2.63 × 10⁴ 0.2179 CT (10) 1.60 × 10⁴ 5.72 ×10⁴ 1.09 × 10⁵ ^(a)The limit of detection for CFU/g or /ml determinationis 1.00 × 10². ^(b)Significance was determined using the one-tailedMann-Whitney test. ^(c)Cholera toxin adjuvant control.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A method of inducing an immune response, comprising administering acomposition comprising an effective amount of at least a portion of oneor more isolated antigens selected from the group consisting of Iha,IreA, IutA and c2482 to a subject under conditions such that saidsubject generates an immune response to E. coli in said subject'surinary tract.
 2. The method of claim 1, wherein said compositionfurther comprises an adjuvant.
 3. The method of claim 2, wherein saidadjuvant is cholera toxin.
 4. The method of claim 3, wherein saidcholera toxin is crosslinked to said antigen.
 5. The method of claim 1,wherein said immune response protects said subject from developingsymptoms of a urinary tract infection.
 6. The method of claim 1, whereinsaid subject exhibits decreased levels of bacteria in said subject'sbladder or kidney.
 7. The method of claim 1, wherein said at least aportion is a peptide that includes extracellular loop 6 of IutA.
 8. Amethod of preventing urinary tract infections by E. coli in a subject,comprising administering a composition comprising an effective amount ofat least a portion of one or more isolated antigens selected from thegroup consisting of Iha, IreA, IutA and c2482 to a subject underconditions such that said subject does not develop symptoms of a urinarytract infection caused by E. coli.
 9. The method of claim 8, whereinsaid subject exhibits decreased levels of bacteria in said subject'sbladder or kidney.
 10. The method of claim 9, wherein said bacteria isE. coli.
 11. The method of claim 8, wherein said composition furthercomprises an adjuvant.
 12. The method of claim 11, wherein said adjuvantis cholera toxin.
 13. The method of claim 12, wherein said cholera toxinis crosslinked to said antigen.
 14. The method of claim 8, wherein saidat least a portion is a peptide that includes extracellular loop 6 ofIutA.
 15. The method of claim 1, wherein said composition furthercomprises an effective amount of at least a portion of IroN.
 16. Themethod of claim 15, wherein said at least a portion of IroN is a peptidethat includes extracellular loop 7 or IronN.
 17. The method of claim 8,wherein said composition further comprises an effective amount of atleast a portion of IroN.
 18. The method of claim 17, wherein said atleast a portion of IroN is a peptide that includes extracellular loop 7or IronN.